Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/06—Tripeptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
  • A61K38/212—IFN-alpha
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0802—Tripeptides with the first amino acid being neutral
  • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
  • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to compounds, processes for their synthesis, compositions and methods for the treatment of hepatitis C virus (HCV) infection,
• HCV hepatitis C virus
• the present invention provides novel peptide analogs, pharmaceutical compositions containing such analogs and methods for using these analogs in the treatment of HCV infection.
• Hepatitis C virus is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 200 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so-called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
• HCV The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protection against HCV infection and disease have yet to be established. Immunoglobulins have been reported for prophylaxis of transfusion-associated viral hepatitis, however, the Center for Disease Control does not presently recommend immunoglobulin treatment for this purpose. The lack of an effective protective immune response is hampering the development of a vaccine or adequate post-exposure prophylaxis measures, so in the near-term, hopes are firmly pinned on antiviral interventions.
• interferon was the only available therapy of proven benefit approved in the clinic for patients with chronic hepatitis C.
• the sustained response rate is low, and interferon treatment also induces severe side-effects (i.e. retinopathy, thyroiditis, acute pancreatitis, depression) that diminish the quality of life of treated patients.
• interferon in combination with ribavirin has been approved for patients non-responsive to IFN alone.
• the side effects caused by IFN are not alleviated with this combination therapy.
• Pegylated forms of interferons such as PEG-Intron® and Pegasys® can apparently partially address these deleterious side-effects but antiviral drugs still remain the avenue of choice for oral treatment of HCV...
• HCV is an enveloped positive strand RNA virus in the Flaviviridae family.
• the single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins.
• NS structural and non-structural
• the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A. and NS5B) is effected by two viral proteases.
• the first one cleaves at the NS2-NS3 junction (henceforth referred to as NS2/3 protease); the second one is a serine protease contained within the N-terminal region of NS3 (NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites.
• the NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components.
• NS3 protease The complex formation of the NS3 protease with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites.
• the NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities.
• NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
• a general strategy for the development of antiviral agents is to inactivate virally encoded enzymes that are essential for the replication of the virus.
• the NS3 protease has been found to potentially have an additional impact by blocking the IFN-mediated cellular antiviral activity in the infected cell ( Foy et al., Science, 17 April 2003 ). This lends credence to a hypothesis that the NS3/NS4A protease may represent a dual therapeutic target, the inhibition of which may both block viral replication and restore interferon response of HCV infected cells.
• hepatitis C viral NS3 protease inhibitors an enzyme essential for the replication of the hepatitis C virus.
• the present invention provides tripeptide compounds that have improved potency against the HCV NS3 protease. Furthermore, compounds being highly active in cell culture are provided.
• An advantage of one aspect of the present invention resides in the fact that compounds according to this invention specifically inhibit the NS3 protease and do not show significant inhibitory activity against other human serine proteases such as human leukocyte elastase (HLE), or cysteine proteases such as human liver cathepsin B (Cat B).
• HLE human leukocyte elastase
• Cat B human liver cathepsin B
• composition comprising an anti-hepatitis C virally effective amount of a compound of formula I , or a pharmaceutically acceptable salt or ester thereof, in admixture with at least one pharmaceutically acceptable carrier medium or auxiliary agent.
• the pharmaceutical composition according to this invention further comprises a therapeutically effective amount of at least one other antiviral agent.
• Another important aspect of the invention involves a method of treating or preventing a hepatitis C viral infection in a mammal by administering to the mammal an anti-hepatitis C virally effective amount of a compound of formula I, a pharmaceutically acceptable salt or ester thereof, or a composition as described above, alone or in combination with at least one other antiviral agent, administered together or separately.
• a compound of formula I or a pharmaceutically acceptable salt or ester thereof, as described herein, for the manufacture of a medicament for the treatment or prevention of hepatitis C viral infection in mammal.
• P1, P2, and P3 refer to the position of the amino acid residues starting from the C-terminus end of the peptide analogs and extending towards the N-terminus (i.e. P1 refers to position 1 from the C-terminus, P2: second position from the C-terminus, etc.) (see Berger A. & Schechter I., Transactions of the Royal Society London series B257, 249-264 (1970 )).
• (1 R , 2 S )-viny)-ACCA refers to a compound of formula: namely, (1 R , 2 S ) 1-amino-2-ethenylcyclopropanecarboxylic acid.
• (C 1-n )alkyl as used herein, either alone or in combination with another substituent, means acyclic, straight or branched chain alkyl substituents containing from 1 to n carbon atoms.
• “(C 1-6 )alkyl” includes, but is not limited to, methyl, ethyl, n-propyl, n-butyl, 1-methylethyl (i-propyl), 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl ( tert -butyl), pentyl and hexyl.
• Me and Pr denote a methyl group and n-propyl respectively.
• (C 3-7 )cycloalkyl as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent containing from 3 to 7 carbon atoms and includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
• (C 1-n )alkyl-(C 3-7 )cycloalkyl as used herein means an alkylene radical containing 1 to n carbon atoms to which a cycloalkyl radical containing from 3 to 7 carbon atoms is directly linked;and includes, but is not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, 1-cyclopentylethyl, 2-cyclopentylethyl, cyclohexylmethyl, 1-cyclohexylethyl, 2-cyclohexylethyl and cycloheptylpropyl.
• aryl or "C 6 or C 10 aryl” as used herein interchangeably, either alone or in combination with another radical, means either an aromatic monocyclic group containing 6 carbon atoms or an aromatic bicyclic group containing 10 carbon atoms.
• Aryl includes, but is not limited to, phenyl, 1-naphthyl or 2-naphthyl.
• (C 1-n )alkyl-aryl means an alkyl radical containing from 1 to n carbon atoms to which an aryl is bonded.
• Examples of (C 1-3 )alkyl-aryl include, but are not limited to, benzyl (phenylmethyl), 1-phenylethyl, 2-phenylethyl and phenylpropyl.
• O-(C 1-n )alkyl or "(C 1-n )alkoxy” as used herein, either alone or in combination with another radical, means the radical -O-(C 1-n )alkyl wherein alkyl is as defined above containing from 1 to n carbon atoms, and includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy.
• alkyl is as defined above containing from 1 to n carbon atoms, and includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy.
• the latter radical is known commonly as tert -butoxy.
• halo or halogen as used herein means a halogen substituent selected from fluoro, chloro, bromo or iodo.
• esters of the compound of formula I in which any of the carboxyl functions of the molecule, but preferably the carboxy terminus, is replaced by an alkoxycarbonyl function: in which the R moiety of the ester is selected from alkyl (including, but not limited to, methyl, ethyl, n -propyl, t-butyl, n -butyl); alkoxyalkyl (including, but not limited to methoxymethyl); alkoxyacyl (including, but not limited to acetoxymethyl); alkyl-aryl (including, but not limited to benzyl); aryloxyalkyl (including, but not limited to phenoxymethyl); aryl (including, but not limited to phenyl), optionally substituted with halogen, (C 1-4 )alkyl or (C 1-4 )alkoxy.
• alkyl including, but not limited to, methyl, ethyl, n -propyl, t-butyl, n
• any alkyl moiety present advantageously contains 1 to 16 carbon atoms, particularly 1 to 6 carbon atoms.
• Any aryl moiety present in such esters advantageously comprises a phenyl group.
• esters may be a C 1-16 alkyl ester, an unsubstituted benzyl ester or a benzyl ester substituted with at least one halogen, C 1-6 alkyl, C 1-6 alkoxy, nitro or trifluoromethyl.
• pharmaceutically acceptable salt means a salt of a compound of formula (I) which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, generally water or oil-soluble or dispersible, and effective for their intended use.
• pharmaceutically-acceptable acid addition salts and pharmaceutically-acceptable base addition salts. Lists of suitable salts are found in, e.g., S.M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19 .
• pharmaceutically-acceptable acid addition salt means those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoric acid, hemisulfic acid, hexanoic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid
• pharmaceutically-acceptable base addition salt means those salts which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable, formed with inorganic bases such as ammonia or hydroxide, carbonate, or bicarbonate of ammonium or a metal cation such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
• Salts derived from pharmaceutically-acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, quaternary amine compounds, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion-exchange resins, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium
• mammal as it is used herein is meant to encompass humans, as well as non-human mammals which are susceptible to infection by hepatitis C virus including domestic animals, such as cows, pigs, horses, dogs and cats, and non-domestic animals.
• antiviral agent means an agent (compound or biological) that is effective to inhibit the formation and/or replication of a virus in a mammal. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of a virus in a mammal. Such agents can be selected from: another anti-HCV agent, HIV inhibitor, HAV inhibitor and HBV inhibitor.
• Antiviral agents include, for example, ribavirin, amantadine, VX-497 (merimepodib, Vertex Pharmaceuticals), VX-498 (Vertex Pharmaceuticals), Levovirin, Viramidine, Ceplene (maxamine), XTL-001 and XTL-002 (XTL Biopharmaceuticals).
• other anti-HCV agent means those agents that are effective for diminishing or preventing the progression of hepatitis C related symptoms of disease.
• agents can be selected from: immunomodulatory agents, inhibitors of HCV NS3 protease, inhibitors of HCV polymerase or inhibitors of another target in the HCV life cycle.
• immunomodulatory agent means those agents (compounds or biologicals) that are effective to enhance or potentiate the immune system response in a mammal.
• Immunomodulatory agents include, for example, class I interferons (such as ⁇ -, ⁇ -, ⁇ -, ⁇ - and ⁇ -interferons, consensus interferons and asialo-interferons), class II interferons (such as ⁇ -interferons) and pegylated forms thereof.
• inhibitor of HCV NS3 protease means an agent (compound or biological) that is effective to inhibit the function of HCV NS3 protease in a mammal.
• Inhibitors of HCV NS3 protease include, for example, those compounds described in WO 99/07733 , WO 99/07734 , WO 00/09558 , WO 00/09543 , WO 00/59929 , WO 03/064416 , WO 03/064455 , WO 03/064456 , WO 02/060926 , WO 03/053349 , WO 03/099316 or WO 03/099274 , and the Vertex pre-development candidate identified as VX-950.
• inhibitor of HCV polymerase means an agent (compound or biological) that is effective to inhibit the function of an HCV polymerase in a mammal. This includes, for example, inhibitors of HCV NS5B polymerase. Inhibitors of HCV polymerase include non-nucleosides, for example, those compounds described in:
• inhibitor of another target in the HCV life cycle means an agent (compound or biological) that is effective to inhibit the formation and/or replication of HCV in a mammal other than by inhibiting the function of the HCV NS3 protease. This includes agents that interfere with either host or HCV viral mechanisms necessary for the formation and/or replication of HCV in a mammal.
• Inhibitors of another target in the HCV life cycle include, for example, agents that inhibit a target selected from helicase, NS2/3 protease and internal ribosome entry site (IRES).
• ISIS-14803 ISIS Pharmaceuticals.
• HIV inhibitor means an agents (compound or biological) that is effective to inhibit the formation and/or replication of HIV in a mammal. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of HIV in a mammal. HIV inhibitors include, for example, nucleoside inhibitors, non-nucleoside inhibitors, protease inhibitors, fusion inhibitors and integrase inhibitors.
• HAV inhibitor means an agent (compound or biological) that is effective to inhibit the formation and/or replication of HAV in a mammal. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of HAV in a mammal.
• HAV inhibitors include Hepatitis A vaccines, for example, Havrix ® (GlaxoSmithKline), VAQTA ® (Merck) and Avaxim ® (Aventis Pasteur).
• HBV inhibitor means an agent (compound or biological) that is effective to inhibit the formation and/or replication of HBV in a mammal. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of HBV in a mammal.
• HBV inhibitors include, for example, agents that inhibit HBV viral DNA polymerase or HBV vaccines.
• HBV inhibitors include Lamivudine (Epivir-HBV ® ), Adefovir Dipivoxil, Entecavir, FTC (Coviracil ® ), DAPD (DXG), L-FMAU (Clevudine ® ), AM365 (Amrad), Ldt (Telbivudine), monoval-LdC (Valtorcitabine), ACH-126,443 (L-Fd4C) (Achillion), MCC478 (Eli Lilly), Racivir (RCV), Fluoro-L and D nucleosides, Robustaflavone, ICN 2001-3 (ICN), Bam 205 (Novelos), XTL-001 (XTL), Imino-Sugars (Nonyl-DNJ) (Synergy), HepBzyme; and immunomodulator products such as: interferon alpha 2b, HE2000 (Hollis-Eden), Theradigm (E
• class I interferon means an interferon selected from a group of interferons that all bind to receptor type I. This includes both naturally and synthetically produced class I interferons. Examples of class I interferons include ⁇ -, ⁇ -, ⁇ -, ⁇ - and ⁇ -interferons, consensus interferons, asialo-interferons and pegylated forms thereof.
• class II interferon as used herein means an interferon selected from a group of interferons that all bind to receptor type II. Examples of class II interferons include ⁇ -interferons.
• combination therapy is contemplated wherein a compound of formula (I), or a pharmaceutically acceptable salt thereof, is co-administered with at least one additional agent selected from: an antiviral agent, an immunomodulatory agent, another inhibitor of HCV NS3 protease, an inhibitor of HCV polymerase, an inhibitor of another target in the HCV life cycle, an HIV inhibitor, an HAV inhibitor and an HBV inhibitor.
• additional agents may be combined with the compounds of this invention to create a single pharmaceutical dosage form. Alternatively these additional agents may be separately administered to the patient as part of a multiple dosage form, for example, using a kit. Such additional agents may be administered to the patient prior to, concurrently with, or following the administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• treatment means the administration of a compound or composition according to the present invention to alleviate or eliminate symptoms of the hepatitis C disease and/or to reduce viral load in a patient.
• prevention means the administration of a compound or composition according to the present invention post-exposure of the individual to the virus but before the appearance of symptoms of the disease, and/or prior to the detection of the virus in the blood, to prevent the appearance of symptoms of the disease.
• the designation whereby a bond to a substituent R is drawn as emanating from the center of a ring, such as, for example, means that the substituent R may be attached to any free position on the ring that would otherwise be substituted with a hydrogen atom, unless specified otherwise.
• B is preferably selected from (C 2-8 )alkyl, (C 3-7 )cycloalkyl and (C 1-3 )alkyl-(C 3-7 )cycloalkyl,
• B is selected from ethyl, n-propyl, i-propyl, n-butyl, 1-methylpropyl, 2-methylpropyl, tert -butyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl,
• B is even more preferably selected from ethyl, 1-methylethyl, 1,1-dimethylethyl, propyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylpropyl, 1-ethyl-2-methylpropyl, 1-(1-methylethyl)-2-methylpropyl, 1-ethyl-2,2-dimethylpropyl, butyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,2-dimethylbutyl, 1,1-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1,2,2-trimethylbutyl, 1,2,3-trimethylbutyl, 2,2,3-trimethylbuty
• B is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl or is selected from the following formulas, wherein one or two CH 2 -groups of a cycloalkyl group is replaced by oxygen:
• cycloalkyl and alkyl-cycloalkyl groups optionally comprising 1 or 2 O-atoms are optionally substituted with 1, 2 or 3 methyl-groups.
• cycloalkyl groups, optionally comprising 1 or 2 O-atoms are preferred, wherein the ⁇ -C-atom is substituted with methyl.
• B is selected from ethyl, n-propyl, tert -butyl, 2-methylpropyl, 1,2-dimethylpropyl, 1,2,2-trimethylpropyl, 2-fluoroethyl, 3-fluoropropyl, 3,3,3-trifluoropropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl and 1-methylcyclohexyl, and a group selected from:
• B is selected from ethyl, n -propyl, tert -butyl, cyclopentyl, 1-methylcyclopentyl, 2-fluoroethyl or 3-fluoropropyl.
• X is O.
• X is NH
• R 3 is preferably (C 2-6 )alkyl, (C 3-7 )cycloalkyl or (C 1-3 )alkyl-(C 3-7 )cycloalkyl, each of which being optionally substituted with 1 to 3 substituents selected from (C 1-4 )alkyl.
• R 3 is more preferably selected from ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, or cyclohexylmethyl, each of which optionally being substituted with 1 or 2 substituents selected from methyl, ethyl and propyl.
• R 3 is selected from 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl, 1-methylcyclohexyl, cyclopentylmethyl, cyclohexylmethyl, (1-methylcyclopentyl)methyl and (1-methylcyclohexyl)methyl.
• R 3 is most preferably selected from 1,1-dimethylethyl, cyclopentyl, cyclohexyl and 1-methylcyclohexyl.
• R 3 is most preferably selected from 1,1-dimethylethyl and cyclohexyl.
• L 0 is preferably selected from H, halogen, CH 3 , -OH, -OCH 3 , -OC 2 H 5 , -OC 3 H 7 , -OCH(CH 3 ) 2 , -NHCH 3 , -NHC 2 H 5 , -NHC 3 H 7 , -NHCH(CH 3 ) 2 , -N(CH 3 ) 2 , -N(CH 3 )C 2 H 5 , -N(CH 3 )C 3 H 7 and -N(CH 3 )CH(CH 3 ) 2 .
• L 0 is selected from H, -OH, -OCH 3 , halogen and -N(CH 3 ) 2 .
• L 0 is H, -OH or-OCH 3 .
• L 0 is H or -OCH 3 .
• L 1 and L 2 are preferably each independently selected from: halogen, -CH 3 , -C 2 H 5 , -OCH 3 , -OC 2 H 5 , -OC 3 H 7 , -OCH(CH 3 ) 2 , CF 3 , -SMe, -SOMe, and SO 2 Me whereby either L 1 or L 2 may be H.
• L 1 and L 2 are -CH 3 , -F, -Cl, -Br, -OMe, -SMe, or -SO 2 Me and the other of L 1 and L 2 is H.
• L 1 is CH 3 , -F, -Cl, -Br, -OMe, -SMe, or -SO 2 Me and L 2 is H.
• L 0 is selected from: H, -OH and -OCH 3 ; and either one of L 1 and L 2 is CH 3 , -F, -Cl, -Br, -OMe, -SMe, or -SO 2 Me and the other of L 1 and L 2 is H.
• L 0 is selected from H, -OH and -OCH 3 ;
• L 1 is - CH 3 , -F, -Cl, -Br, -OMe, -SMe, or -SO 2 Me; and
• L 2 is H.
• L 0 is H or -OCH 3 ;
• L 1 is -CH 3 , Cl or Br, and
• L 2 is H.
• this ring system is preferably selected from: wherein
• this ring system is preferably selected from: wherein
• L 0 and L 1 are covalently bonded to form, together with the quinoline residue to which they are linked, a ring system which is selected from: wherein each R b is independently (C 1-4 )alkyl and L 2 is as defined; preferably H or methyl, particularly H.
• L 0 and L 1 are covalently bonded to form together with the quinoline residue to which they are linked a ring system selected from wherein L 2 is H or -CH 3 , preferably H.
• R 2 is preferably R 20 , -NHCO R 20 , -NHCOO R 20 , -NH R 21 and -NHCON R 21 R 23 , wherein R 20 is selected from (C 1-8 )alkyl, (C 3-7 )cycloalkyl, (C 1-3 )alkyl-(C 3-7 )cycloalkyl, wherein said cycloalkyl and alkyl-cycloalkyl may be mono-, di- or tri-substituted with (C 1-3 )alkyl; and R 21 is H or R 20 as defined above; and R 23 is H or methyl; most preferably H.
• R 2 is R 20 , NHCO R 20 , -NHCOO R 20 , -NH R 21 -and -NHCON R 21 R 23 , wherein R 20 is selected from methyl, ethyl, n -propyl, i -propyl, n -butyl, 1-methylpropyl, 2-methylpropyl, tert -butyl, 2,2-dimethylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1,2,2-trimethylpropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl, each of said cycloalkyl and alkyl-cycloalkyl groups being optionally substituted with 1 to 3 substituents selected from methyl and ethyl, in particular methyl; and
• R 2 is selected from:
• R 2 is -NHCO R 20 , -NHCOO R 20 , or -NH R 21 , wherein R 20 and R 21 are as defined herein.
• R 20 and R 21 are independently selected from: methyl, ethyl, n -propyl, i- propyl, n -butyl, 1-methylpropyl, 2-methylpropyl, tert -butyl, 2,2-dimethylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1,2,2-trimethylpropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, each of said cycloalkyl or alkyl-cycloalkyl groups optionally being mono- or di-substituted with methyl or ethyl.
• R 20 and R 21 are independently selected from: methyl, ethyl, n- propyl, i -propyl, 2,2-dimethylpropyl, cyclopentyl and cyclopentylmethyl.
• R 1 is vinyl
• the asymmetric carbon atoms in the cyclopropyl group take the R,S configuration according to the subformula:
• R 1 is preferably vinyl.
• R C is preferably selected from hydroxy or NHSO 2 R S wherein R S is methyl, ethyl, n -propyl, i -propyl, n -butyl, 1-methylpropyl, 2-methylpropyl, tert -butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, phenyl, naphthyl, pyridinyl, phenylmethyl (benzyl), naphthylmethyl or pyridinylmethyl;
• R C is NHSO 2 R S , wherein R S is -N( R N2 ) R N1 ), wherein R N1 and R N2 are independently selected from H, (C 1-4 )alkyl, (C 3-7 )cycloalkyl, (C 1-3 )alkyl-(C 3-7 )cycloalkyl, phenyl, and (C 1-3 )alkyl-phenyl; wherein said (C 1-4 )alkyl, (C 3-7 )cycloalkyl, (C 1-3 )alkyl-(C 3-7 )cycloalkyl, phenyl and (C 1-3 )alkyl-phenyl are optionally substituted with one, two or three substituents independently selected from halogen, (C 1-6 )alkyl, hydroxy, cyano, O-(C 1-6 )alkyl, -NH 2 , -NH(C 1-4 )alkyl, -N((C 1-4
• the group R C is hydroxy, NHSO 2 -methyl, NHSO 2 -ethyl, NHSO 2 -(1-methyl)ethyl, NHSO 2 -propyl, NHSO 2 -cyclopropyl, NHSO 27 CH 2 -cyclopropyl, NHSO 2 -cyclobutyl, NHSO 2 -cyclopentyl, or NHSO 2 -phenyl.
• R C is hydroxy, or NHSO 2 -cyclopropyl.
• the group R C is hydroxy. According to an alternative most preferred embodiment, the group R C is NHSO 2 -cyclopropyl. According to another alternative most preferred embodiment, the group R C is NHSO 2 N(CH 3 ) 2 .
• B is selected from: ethyl, n-propyl, tert -butyl, 2-methylpropyl, 1,2-dimethylpropyl, 1,2,2-trimethylpropyl, 2-fluoroethyl, 3-fluoropropyl, 3,3,3-trifluoropropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl and 1-methylcyclohexyl, and a group selected from:
• R 3 is selected from 1,1-dimethylethyl, cyclopentyl, cyclohexyl and 1-methylcyclohexyl;
• L 0 is H, -OH or-OCH 3 ;
• L 1 is CH 3 , -F, -Cl, -Br, -OMe, -SMe, or -SO 2 Me;
• L 2 is H;
• B is selected from ethyl, n -propyl, tert -butyl, cyclopentyl, 1-methylcyclopentyl, 2-fluoroethyl and 3-fluoropropyl;
• R 3 is selected from 1,1-dimethylethyl and cyclohexyl;
• L 0 is H or -OCH 3 ;
• L 1 is -CH 3 , -Cl, or -Br;
• L 2 is H; and
• R C is hydroxy.
• Examples of preferred embodiments according to this invention is each single compound listed in the following Tables 1, 2, 3, 4, 5 and 6; 6 and 7.
• the pharmaceutical composition of this invention may additionally comprise at least one other anti-HCV agent.
• anti-HCV agents include, ⁇ - (alpha), ⁇ - (beta), ⁇ - (delta), ⁇ - (gamma), ⁇ - (omega) or ⁇ - (tau) interferon, pegylated ⁇ -interferon, ribavirin and amantadine.
• the pharmaceutical composition of this invention may additionally comprise at least one other inhibitor of HCV NS3 protease.
• the pharmaceutical composition of this invention may additionally comprise at least one inhibitor of HCV polymerase.
• the pharmaceutical composition of this invention may additionally comprise at least one inhibitor of other targets in the HCV life cycle, including but not limited to, helicase, NS2/3 protease or internal ribosome entry site (IRES).
• at least one inhibitor of other targets in the HCV life cycle including but not limited to, helicase, NS2/3 protease or internal ribosome entry site (IRES).
• the pharmaceutical composition of this invention may be administered orally, parenterally or via an implanted reservoir. Oral administration or administration by injection is preferred.
• the pharmaceutical composition of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
• the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
• parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, and intralesional injection or infusion techniques.
• the pharmaceutical composition may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
• This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example Tween 80) and suspending agents.
• the pharmaceutical composition of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions.
• carriers which are commonly used include lactose and corn starch.
• Lubricating agents such as magnesium stearate, are also typically added.
• useful diluents include lactose and dried corn starch.
• aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
• Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, preferably between about 0.1 and about 50 mg/kg body weight per day of the protease inhibitor compound described herein are useful in a monotherapy for the prevention and treatment of HCV mediated disease.
• the pharmaceutical composition of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
• a typical preparation will contain from about 5% to about 95% active compound ( w / w ).
• such preparations contain from about 20% to about 80% active compound.
• composition of this invention comprises a combination of a compound of formula I and one or more additional therapeutic or prophylactic agent
• both the compound and the additional agent should be present at dosage levels of between about 10 to 100%, and more preferably between about 10 and 80% of the dosage normally administered in a monotherapy regimen.
• compositions may be administered in vivo to mammals, such as man, to ' inhibit HCV NS3 protease or to treat or prevent HCV virus infection. Such treatment may also be achieved using a compound of this invention in combination with another antiviral agent.
• Preferred other antiviral agents are described within the Definitions section and the section of preferred pharmaceutical compositions according to this invention and include, but are not limited to: ⁇ -, ⁇ -, ⁇ -, ⁇ -, ⁇ - or ⁇ -interferon, ribavirin, amantadine; other inhibitors of HCV NS3 protease; inhibitors of HCV polymerase; inhibitors of other targets in the HCV life cycle, which include but are not limited to, helicase, NS2/3 protease, or internal ribosome entry site (IRES); or combinations thereof.
• the additional agents may be combined with compounds of this invention to create a single dosage form. Alternatively these additional agents may be separately administered to a mammal as part of a multiple dosage form.
• another embodiment of this invention provides a method of inhibiting HCV NS3 protease activity in a mammal by administering a compound of the formula I, including a pharmaceutically acceptable salt or ester thereof.
• this method is useful in decreasing the NS3 protease activity of the hepatitis C virus infecting a mammal.
• combination therapy is contemplated wherein a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, is co-administered with at least one additional antiviral agent.
• additional antiviral agents are described hereinbefore and examples of such agents are provided in the Definitions section.
• These additional agents may be combined with the compounds of this invention to create a single pharmaceutical dosage form. Alternatively these additional agents may be separately administered to the patient as part of a multiple dosage form, for example, using a kit. Such additional agents may be administered to the patient prior to, concurrently with, or following the administration of a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof.
• a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, set forth herein may also be used as a laboratory reagent.
• a compound of this invention, including a pharmaceutically acceptable salt or ester thereof may also be used to treat or prevent viral contamination of materials and therefore reduce the risk of viral infection of laboratory or medical personnel or patients who come in contact with such materials (e.g. blood, tissue, surgical instruments and garments, laboratory instruments and garments, and blood collection apparatuses and materials).
• a compound of formula (I), including a pharmaceutically acceptable salt or ester thereof, set forth herein may also be used as a research reagent.
• a compound of formula (I), including a pharmaceutically acceptable salt or ester thereof, may also be used as positive control to validate surrogate cell-based assays or in vitro or in vivo viral replication assays.
• dipeptide 3 is carried out by coupling the P1 residue to the properly protected trans -hydroxy proline under standard conditions.
• the stereochemistry of the hydroxyl group is inverted by the well known Mitsunobu reaction using para -nitrobenzoic acid.
• Coupling of dipeptide with the P3 moiety yielded tripeptide 6 .
• introduction of the quinoline moiety to the hydroxyl group of the tripeptide 7 with inversion of stereochemistry can be carried out using either a Mitsunobu reaction or by converting the free hydroxyl group into a good leaving group (such as a brosylate) and displacing it with the hydroxyl quinoline derivative 9 .
• the quinoline used contains a 2-carbomethoxy group as shown in 9 .
• Conversion of the carboxylate group to the aminothiazole derivative is carried out by well known synthetic methodology and is described and exemplified in WO 00/09543 and WO 00/09598 .
• the C-terminal ester is hydrolyzed under basic aqueous conditions.
• Scheme 2 describes another reaction sequence for making compounds of Formula I.
• the quinoline moiety is introduced to the dipeptide in a similar way as described in Scheme 1.
• the P3 moiety is coupled under standard conditions to the dipeptide 21 . Conversion of the resulting tripeptide to the final compound is carried out as described in Scheme 1.
• P1 moieties of compounds of Formula (I) were prepared using the protocols outlined in WO 00/59929, published October 12, 2000 , and WO 00/09543 , published on February 24, 2000. In particular, reference is made to pages 33-35, Example 1 of WO00/59929 and Pages 56-69, Example 9-20 of WO00/09543 for the preparation of 1-aminocyclopropanecarboxylic acid P1 moieties.
• property substituted anilines at the 2, 3 and/or 4 position are allowed to react with dimethyl acetylene dicarboxylate and the resulting enamine is heated at high temperatures to effect the cyclization.
• the corresponding anilines are commercially available or may require some well known chemical transformation.
• the nitro is commercially available and is then converted to the corresponding amine by using a reducing agent.
• the carboxylic acid when commercially available, it can be transformed into the corresponding amine via a Curtius rearrangement.
• the reaction was rendered basic by the addition of 1 N NaOH (25 mL - final pH >10).
• the solution was washed with EtOAc (2x 200 mL) and the aqueous phase acidified with 1 N HCl (ca. 70 mL - final pH ⁇ 2).
• the turbid solution was extracted with EtOAc (200 + 150 mL).
• the extract was dried (MgSO 4 ) and evaporated to give carbamate 4a as a white solid (8.68 g).
• the reaction mixture was diluted with EtOAc, washed with 10% citric acid (2x), water (2x), saturated NaHCO 3 (2x), water (2x) and brine (1x), dried (MgSO 4 ), filtered and evaporated to obtain the crude compound as a nearly colorless oil (3.73 g; >100%; assume 9.89 mmol).
• the crude product (1.01 g; 2.97 mmol) was dissolved in DMSO (6.5 mL) and cyclopentylamine was added dropwise. The reaction mixture was stirred at R.T. for 45 min and then diluted with EtOAc.
• the organic phase was washed with 10% citric acid (2x), water (2x), saturated NaHCO 3 (2x), water (2x) and brine (1x), dried (MgSO 4 ), filtered and evaporated to give the crude cyclopentyl urea -Tbg-OBn product as a nearly colorless oil.
• the crude material was purified by flash column chromatography with silica using hexane:EtOAc 9:1 to remove the less polar impurities and 7:3 to elute the purified product as a thick colorless oil (936 mg; 95%).
• the benzyl ester product (936 mg; 2.82 mmol) was deprotected under a hydrogen filled balloon at R.T.
• N-tert -butyl- N' -cyclopentylthiourea as a white solid (3.70 g; 47% yield).
• the N-tert -butyl- N' -cyclopentylthiourea (3.70 g) was dissolved in concentrated HCl (46 mL). The dark yellow solution was heated at a gentle reflux. After 40 min the reaction mixture was allowed to cool to R.T. and thereafter cooled in ice and rendered basic to pH 9.5 with solid and a saturated aqueous solution of NaHCO 3 . The product was extracted into EtOAc (3x).
• the diester A4 (6.5 g, 23.27 mmol) was dissolved in diphenyl ether (12 mL) and the reaction mixture placed into a pre-heated sand bath at a bath temperature of 350-400°C. Once the reaction mixture attained an internal temperature of 240°C (observe MeOH evolution at 230-240*C) a count of six minutes was begun before the bath (temperature end point: 262°C) was removed and the reaction allowed to cool to room temperature. A solid formed upon cooling which was diluted with ether, filtered and dried to give a tan brown solid (3.48 g crude).
• the nitrophenol starting material B2 (3.1 g; 14.2 mmol) was dissolved in DMF (20 mL) and to the solution was added ground cesium carbonate (5.58 g; 17.1 mmol) followed by Mel (2.6 mL; 42.5 mmol). The mixture was stirred at room temperature overnight. The DMF was evaporated, the residue taken up in ether (1x 200 mL), washed with water (1 x 200 mL), brine (4x 100 mL), dried (MgSO 4 ), filtered and evaporated to afford the crude 2-bromo-3-nitroanisole B3 (94%; 3.1 g) as an orange solid. MS 234 (M+2H) + ; Homogeneity by HPLC (TFA) @ 220 nm: 98%.
• This diazonium intermediate was poured into a solution of Cu(I)Cl (3.8 g; 38.9 mmol) in H 2 O (18.5 mL) and conc. HCl (18.5 mL) at 0°C. The reaction was stirred for 15 min at 0°C, warmed to 60°C, and stirred for an additional 15 min The reaction mixture was then brought to room temperature, and left to stir overnight. The reaction mixture was transferred to a separatory funnel and extracted with ether (3X 150 mL). The organic layers were combined, washed with brine (1X), dried (Na 2 SO 4 ), filtered and concentrated to afford the crude product (5.83 g) as a red-brown oil.
• the nitrophenol starting material C2 (1.3 g; 7.49 mmol) was dissolved in DMF (10 mL) and to this solution was added ground cesium carbonate (2.92 g; 8.96 mmol), followed by Mel (1.4 mL; 22.5 mmol). The mixture was stirred at room temperature overnight. The DMF was evaporated in vacuo and the residue taken up in ether (150 mL), washed with water (150 mL), brine (4x 100 mL), and then dried over (MgSO 4 ): The organic phase was filtered and evaporated to afford the crude 2-chloro-3-nitroanisole C3 (98%; 1.38 g) as an orange solid. Homogeneity by HPLC (TFA) @ 220 nm: 93%.
• the Michael adduct D4 was dissolved in warm diphenyl ether (6 mL) and placed in a sand bath previously heated to ⁇ 350°C. The internal temperature of the reaction was monitored and maintained at ⁇ 245°C for about 5 minutes (solution turns brown). After cooling to R.T., the desired 4-hydroxyquinoline crashed out of solution. The brown solid was filtered and washed several times with diethyl ether to give, after drying, quinoline D5 as a brown solid (0.51 g, 45%). MS: 252 (M + H) + , 249.9 (M - H) - .
• the Michael adduct E7 was dissolved in warm diphenyl ether (10 mL) and placed in a sand bath previously heated to ⁇ 350°C. The, internal temperature of the reaction was monitored, maintained at ⁇ 245°C for about 5 minutes (solution turns brown) and cooled to R.T. at which time the desired 4-hydroxyquinoline precipitated out of solution. The brown solid was filtered and washed several times with diethyl ether to give quinoline E8 as a yellow-brown solid after drying (1.10 g, 88 %).
• the diester F3 (1.8 g, 6.44 mmol) was dissolved in diphenyl ether (5 mL) and the reaction mixture placed into a pre-heated sand bath at a bath temperature of 350-400°C. Once the reaction mixture attained an internal temperature of 240°C, a count of five minutes was begun before the bath was removed and the reaction allowed to cool to room temperature overnight. A solid formed upon cooling which was diluted with ether, filtered and dried to give a brown solid (0.97 g crude) containing both regioisomers in almost equal proportions.
• the Boc starting material G2 (257 mg; 1.08 mmol) was dissolved in 4M HCl/dioxane (5.0 mL) and stirred at room temperature for 1 hr. The solvent was evaporated and the residue diluted with saturated sodium bicarbonate (few mL) and 1 M NaOH (1 mL), extracted with EtOAc (2x), dried (MgSO 4 ), filtered and evaporated to dryness to provide the crude 2,3-methylene dioxyaniline G3 (158 mg; 106%).
• the diester G4 (180 mg, 0.645 mmol) was dissolved in diphenyl ether (2.5 mL) and the reaction mixture placed into a pre-heated sand bath at a bath temperature of 350-400°C. Once the reaction mixture attained an internal temperature of 250°C (observe MeOH evolution at 220-230°C) a count of six minutes was begun before the bath (temperature end point :262°C) was removed and the reaction allowed to cool to room temperature. A solid formed upon cooling which was diluted with ether, filtered and dried to give the crude dioxyquinoline G5 (125 mg; 78 %). Purification was not required and the material was used as such in the following reactions. MS (M + H) + ; 246, and (M - H) - ; 248.1. Homogeneity by HPLC (TFA) @ 220 nm :88%.
• Triethylamine (5.0 mL, 35.6 mmol) was added to a flask containing the amine H1 (2.0 mL, 17.8 mmol) and dichloromethane (100 mL) under an atmosphere of nitrogen. The contents were cooled in an ice bath and trimethylacetylchloride (3.3 mL, 26.7 mmol) was added dropwise. The reaction was allowed to warm slowly to R.T. and stirred for 14 h. at this temperature. The reaction was quenched with NaHCO 3 saturated solution and extracted with EtOAc. The combined organic layers were dried, filtered and concentrated followed by flash column chromatography (4:1 to 1:1 hexane:EtOAc) to yield the desired product H2 as an off-white solid (3.7 g, 97 % yield).
• n-BuLi (15.9 mL, 1.6M, 25.5 mmol) was added dropwise to a flame dried flask containing a solution of the starting amide H2 (1.6 g, 7.72 mmol) in THF at 0°C under argon. Solution turned slight yellow/orange color when n-BuLi was added. The solution was allowed to warm slowly to R.T. and stirred for 24 h. The solution was again cooled to 0°C and ethylene oxide (0.46 mL, 9.26 mmol) was added dropwise.
• the diester J2 (10.53 g, 37.43 mmol) was dissolved in diphenyl ether (35 mL) and the reaction mixture placed into a pre-heated sand bath at a bath temperature of 350-400°C. Once the reaction mixture attained an internal temperature of 245°C, a count of six minutes was begun before the bath was removed and the reaction allowed to cool to room temperature. A precipitate formed, which was suspended in ether, filtered and washed again with ether to provide the C8-SMe quinoline product J3 (6.15 g; 66%). MS (M + H) + ; 250 Homogeneity by HPLC (TFA) @ 220 nm: 99%.
• the diester K5 (1.13 g, 3.52 mmol) was dissolved in diphenyl ether (3.0 mL) and the reaction mixture placed into a pre-heated sand bath at a bath temperature of 400-440°C. Once the reaction mixture attained an internal temperature of 230°C (observe MeOH evolution at 220°C) a count of six minutes was begun before the bath (temperature end point: 242°C) was removed and the reaction allowed to cool to room temperature.
• This quinoline moiety was used for the synthesis of compounds 1032 and 1033 of table 1.
• quinoline K6 was used for the synthesis of compounds 1034, 1035, 1057 and 1058.
• Conversion of the C7- tert -butyl-ether group to an hydroxyl group was done by treatment of the final compound with 50% TFA in dichloromethane for 30 min. at 0°C then for 30 min. at R.T., evaporated to dryness, diluted with water and lyophilized.
• the diester L2 (10 g, 37.70 mmol) was dissolved in diphenyl ether (15 mL) and the reaction mixture placed into a pre-heated sand bath at a bath temperature of 350-400°C. Once the reaction mixture attained an internal temperature of 240°C, a count of six minutes was begun before the bath was removed and the reaction allowed to cool to room temperature. No solid formed upon cooling, therefore, the crude mixture was flash column purified with hexane : EtOAc (6 : 4 to 5 : 5 to remove impurities, then, 2: 8 to complete elution) to provide the C8-OMe quinoline product L3 (4.56 g; 52%). MS (M - H) - ; 231.9 Homogeneity by HPLC (TFA) @ 220 nm: 99%.
• Dipeptide 1 (72.0 g, 203 mmol), triphenylphosphine (63.94 g, 243.8 mmol, 1.2 equiv.) and 4-nitrobenzoic acid (41.08 g, 245.8 mmol, 1.2 equiv) were dissolved in dry THF (1.4 L) The stirred solution was cooled to 0°C under a nitrogen atmosphere. Diethyl azodicarboxylate (38.4 mL, 244 mmol, 1.2 equiv.) was then added dropwise over 45 min and the reaction allowed to warm to R.T. After 4 h, the solvent was evaporated. The residue was divided into four portions.
• Boc-dipeptide ester 2 was obtained as an amorphous white solid after evaporation of the solvents and drying of the residues under high vacuum at 70°C for 1 h (108.1 g, quantitative).
• a solution of 4N hydrogen chloride in dioxane was added to the Boc-dipeptide ester 2 (108 g, 243 mmol) resulting in a colorless solution. The solution was stirred at R.T. for 1 h. The solvent was evaporated and the residue placed under high vacuum for 3 h affording the hydrochloride salt of compound 3 as an amorphous solid. The solid was used as such.
• the tripeptide 6a (15.68 g) was dissolved in THF (200 mL) and water (30 mL) was added. The resulting solution was cooled to 0°C and a solution of lithium hydroxide monohydrate (1.18 g, 28.12 mmol) was added over 3 min with vigorous stirring. After 3 h at 0°C. the excess base was neutralized with 1N HCl (final pH ca . 6) and the THF evaporated, resulting in an aqueous suspension (yellow gum). The mixture was extracted with EtOAc (2x 200 mL) and washed with saturated NaHCO 3 (2x 300 mL). The combined extracts were dried over MgSO 4 and evaporated to yield a pale yellow foam. Flash chromatography of the foam over silica gel using EtOAc as eluent afforded 7a as a white amorphous solid (9.77 g, 91%).
• brosylate 7aBrs 0.5 g; 0.71 mmol
• bromoquinoline B6 (234 mg; 0.75 mmol)
• ground cesium carbonate 56 mg; 0.78 mmol
• the reaction mixture was poured into EtOAc, washed with H 2 O (1X), NaHCO 3 (saturated; 2X), brine (5X), dried, filtered and concentrated to afford the crude product (0.565 g) as an off white solid.
• Tripeptide 10b (149 mg, 0.210 mmol), in 5 mL of a 1:1 mixture of THF-MeOH, was cooled to 0°C for the addition of a 1N NaOH aqueous solution (0.24 mL, 0.240 mmol). The resulting solution was stirred 15 min at 0°C, 1.5 h at R.T. and found to be incomplete by analytical HPLC. Additional 1N NaOH (0.05 mL, 0.05 mmol) was added and the reaction stirred for an additional hour. The mixture was quenched with 1 M HCl, evaporated to near dryness, diluted with water, frozen and lyophilized to provide the acid 11b (crude material used for next step; assume 0.210 mmol). Reverse Phase HPLC Homogeneity (0.06% TFA; CH 3 CN : H 2 O) : 89%.
• Step 4 Synthesis of thiazolyl tripeptide 14b:
• ⁇ -Bromoketone 13b 49 mg, 0.0635 mmol
• N -neopentylthiourea 8a (12 mg; 0.0688 mmol) were dissolved in isopropanol (3 mL) and the yellow solution was heated at 75°C for 1 hour. The solution was allowed to cool to R.T. and evaporated to dryness. This crude material 14b was used for next step (assume 0.0635 mmol).
• M.S.(electrospray) 845.5 (M-H) - 847.5 (M+H) + .
• Reverse Phase HPLC Homogeneity 0.06% TFA; CH 3 CN : H 2 O
• reaction mixture was concentrated, the residue was dissolved in DMSO and the solution was injected on to a Combi-prep HPLC column. The pure fractions were pooled and lyophilized to yield 16.5 mg (42% yield) of compound 4004 as an amorphous white solid (trifluoroacetic acid salt).
• E11-1 Potassium cyanide (1.43 g, 22.0 mmol) was added to a stirred solution of methylcyclopentanol (2.00 g, 20.0 mmol) in glacial acetic acid (1.00 mL) resulting in a thick slurry. To this was added, dropwise, sulfuric acid (3 mL, caution: exothermic) at a rate at which the temperature was maintained at ca. 30-35°C. Additional acetic acid (1 mL) was added to facilitate stirring of the thick paste. The mixture was then heated to 55-60°C for 30 min followed by stirring at ambient temperature for 16 h.
• E11-2 5N Hydrochloric acid (8 mL) was added to a solution of E11-1 (1.50 g, 11.8 mmol) in dioxane (8.0 mL) resulting in some precipitation. Ethanol (4 mL) was then added and the solution heated to reflux for 4 h. The reaction was then cooled, the organic solvents evaporated and the aqueous residue washed with hexane (40 mL): The aqueous layer was then evaporated to dryness (ethanol was used to azeotrope the last traces of water) and the resulting solid was dried under high vacuum to yield the methylcyclopentylamine hydrochloride as a beige solid (1.38 g, 86%).
• E11-4 The E11-3 (6.80 g, 21.2 mmol) was dissolved in dioxane (4 mL) and a solution of 4N HCl in dioxane (30 mL, 120 mmol) added. After stirring at ambient temperature for 2 h, the solvent was evaporated and the residue allowed to stand under a stream of nitrogen resulting in slow solidification. This material was then triturated with hexane (2x 50 mL), filtered, air dried for 30 min then placed under high vacuum for 5 days to afford the hydrochloride salt as a white solid (4.86 g, 89%).
• E11-5 To a stirred, ice cold solution of E11-4 (4.85 g, 18.8 mmol) in tetrahydrofuran (75 mL) was added diisopropylethylamine (8.20 mL, 47.0 mmol) followed by the dropwise addition of phenylchloroformate (2.60 mL, 20.7 mmol) under an argon atmosphere. A thick precipitate formed which, upon vigorous stirring, became a fine suspension.
• the mixture was concentrated to a third of its original volume and then extracted with ethyl acetate (50 mL) and washed with water (40 mL), 0.5 M KHSO 4 (40 mL), 5 % NaHCO 3 (2x 40 mL) and brine (50 mL).
• the organic phase was dried over MgSO 4 and evaporated to yield the phenyl carbamate as a colorless oil which slowly crystallized over a period of days (6.63 g, quantitative).
• E11-6 To a solution of E11-5 (1.00 g, 2.93 mmol) in DMSO (2.00 mL) containing acetonitrile (1.00 mL) was added diisopropylethylamine (817 ⁇ L) followed by the amine E11-2 (477 mg, 3.52 mmol). The reaction was stirred at ambient temperature for 2 h and then heated to 70°C for 45 min The solution was then diluted with ethyl acetate (30 mL), washed with 5% aq K 2 CO 3 (4x 50 mL) and brine (50 mL).
• E11-7 To solution of urea E11-6 (780 mg, 2.25 mmol) in absolute ethanol (10 mL) under an argon atmosphere was added 10% Pd-C catalyst (100 mg). The system was purged three times with H 2 and then stirred vigorously under a hydrogen-balloon. After 3 h, the catalyst was filtered over Celite and the filtrate evaporated. The residue was then dissolved in methanol (ca. 10 mL), filtered through a Millipore Millex 0.45 uM filter and then evaporated to yield the acid E11-7 as a white solid (539 mg, 93%).
• E11-8 The urea E11-7 (239 mg, 0.932 mmol) and TBTU (3.06 mg, 0.979 mmol) were dissolved/suspended in anhydrous dichloromethane (4 mL) and diisopropylethylamine (157 ⁇ L, 0.900 mmol) added. The reaction was stirred at ambient temperature under a nitrogen atmosphere until the solution became nearly homogeneous ( ca . 5 min).
• E11-10 To a stirred, ice cold suspension of sodium salt E11-9 (0.894 mmol) in tetrahydrofuran (10 mL) under an argon atmosphere was added triethylamine (240 ⁇ L, 1.72 mmol) followed by the dropwise addition of isobutyl chloroformate (174 ⁇ L, 1.34 mmol). The resulting suspension was stirred at 0°C for 3 h and a solution of diazomethane in ethyl ether (0.7M, 10 mL, 7 mmol) then added. The yellow suspension was stirred for 30 min at 0°C and then allowed to warm to ambient temperature. After 1 h, nitrogen was bubbled through the suspension for 15 min.
• Urea Acid E13-1 The urea-P3 acid was prepared from tert-butylamine and E11-5 by the same sequence of reactions as described in Example 11.
• Tripeptide ester E13-2 The urea-P3 acid was coupled with the P1-P2 fragment 15b as described in Example 11.
• E14-2 The urea-P3 acid E14-2 was prepared from E11-5 and amine E14-1 by the same sequence of reactions as described in Example 11.
• the final inhibitor was prepared from E14-3 by a sequence of steps identical to that described on Example 11.
• the product of the final saponification was isolated as an amorphous yellow powder (trifluoroacetate salt, 10 mg, 21 %).
• Step 1 Formation of the aminothiazole ring
• a series of 8-mL vials were disposed in a reaction block from an ACT496 synthesizer (from Advanced Chemtech).
• ACT496 synthesizer from Advanced Chemtech
• the thio-derivative (8) of interest (0.0688 mmole)
• the bromoketone (0.0625 mmole)
• isopropanol 500 ⁇ L.
• the closed vials were heated at 70°C for 1 h.
• the solvent was then evaporated using a vacuum centrifuge (SpeedVac) and was co-evaporated with 1,2-dichloroethane.
• the crude products were dried under high vacuum overnight.
• HATU (20 mg, 0.05 mmol) was added to a solution of compound 17A1 (Compound 3004, Table 3; 20 mg, 0.03 mmol) and DIPEA (0.03 mL, 0.16 mmol) in DMF (1.5 mL) at RT.
• the solution was stirred for 1h followed by the addition of DMAP (16 mg, 0.13 mmol) and cyclopropanesulfonamide (7.0 mg, 0.06 mmol). After addition was complete, the mixture was allowed to stir for 15 min and DBU (0.02 mL, 0.14 mmol) was added dropwise.
• Huh7 cells that stably maintain a subgenomic HCV replicon were established as previously described ( Lohman et al., 1999. Science 285: 110-113 ) and designated as the S22.3 cell-line.
• S22.3 cells are maintained in Dulbecco's Modified Earle Medium (DMEM) supplemented with 10% FBS and 1 mg/mL neomycin (Standard Medium). During the assay, DMEM medium supplemented with 10% FBS, containing 0.5% DMSO and lacking neomycin was used (Assay Medium). 16 hours prior to compound addition, S22.3 cells are trypsinized and diluted to 50 000 cells/mL in Standard Medium. 200 ⁇ L (10 000.
• test compound 10 ⁇ L was added to 2 mL of Assay Medium for a final DMSO concentration of 0.5% and the solution was sonicated for 15 min and filtered through a 0.22 ⁇ M Millipore Filter Unit. 900 ⁇ L was transferred into row A of a Polypropylene Deep-Well Titer Plate. Rows B to H contain 400 ⁇ L aliquots of Assay Medium (containing 0.5% DMSO), and are used to prepare serial dilutions (1/2) by transferring 400 ⁇ L from row to row (no compound was included in row H).
• Cell culture medium was aspirated from the 96-well plate containing the S22.3 cells. 175 ⁇ L of assay medium with the appropriate dilution of test compound was transferred from each well of the compound plate to the corresponding well of the cell culture plate (row H was used as the "No inhibition control"). The cell culture plate was incubated at 37°C with 5% CO 2 for 72 h.
• RLT buffer Qiagen®
• 70% ethanol was then added to each microplate well, and mixed by pipetting.
• the RNeasy 96 plate was sealed with tape and the Square-Well Block with the RNeasy 96 plate was loaded into the holder and placed in a rotor bucket of a 4K15C centrifuge. The sample was centrifuged at 6000 rpm ( ⁇ 5600 x g) for 4 min at room temperature. The tape was removed from the plate and 0.8 mL of Buffer RW1 (Qiagen® RNeasy 96 kit) was added to each well of the RNeasy 96 plate. The RNeasy 96 plate was sealed with a new piece of tape and centrifuged at 6000 rpm for 4 min at room temperature.
• the RNeasy 96 plate was placed on top of another clean Square-Well Block, the tape removed and 0.8 mL of Buffer RPE (Qiagen® RNeasy 96 kit) was added to each well of the RNeasy 96 plate.
• the RNeasy 96 plate was sealed with a new piece of tape and centrifuged at 6000 rpm for 4 min at room temperature.
• the tape was removed and another 0.8 mL of Buffer RPE (Qiagen® RNeasy 96 kit) was added to each well of the RNeasy 96 plate.
• the RNeasy 96 plate was sealed with a new piece of tape and centrifuged at 6000 rpm for 10 min at room temperature.
• RNA was quantified on the STORM® system (Molecular Dynamics®) using the RiboGreen® RNA Quantification Kit (Molecular Probes®). Briefly, the RiboGreen reagent was diluted 200-fold in TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA). Generally, 50 ⁇ L of reagent was diluted in 10 mL TE. A Standard Curve of ribosomal RNA was diluted in TE to 2 ⁇ g/mL and pre-determined amounts (100, 50, 40, 20, 10, 5, 2 and 0 ⁇ L) of the ribosomal RNA solution are then transferred in a new 96-well plate (COSTAR # 3997) and the volume was completed to 100 ⁇ L with TE.
• column 1 of the 96-well plate was used for the standard curve and the other wells are used for the RNA samples to be quantified.
• 10 ⁇ L of each RNA sample that was to be quantified was transferred to the corresponding well of the 96-well plate and 90 ⁇ L of TE was added.
• One volume (100 ⁇ L) of diluted RiboGreen reagent was added to each well of the 96-well plate and incubated for 2 to 5 minutes at room temperature, protected from light (a 10 ⁇ L RNA sample in a 200 ⁇ L final volume generates a 20X dilution).
• the fluorescence intensity of each well was measured on the STORM® system (Molecular Dynamics®).
• RNA concentration in the experimental samples was determined from the standard curve and corrected for the 20X dilution.
• Reagents and Materials Product Company Catalog # Storage DEPC Sigma D5758 4°C EDTA Sigma E5134 R.T. Trizma-Base Sigma T8524 R.T. Trizma-HCl Sigma T7149 R.T. Collection Tube Strips Qiagen 19562 R.T. Ribogreen RNA Quantitation Kit Molecular Probe R11490 -20°C Rneasy 96 Kit Qiagen 74183 R.T. Square-Well Blocks Qiagen 19573 R.T.
• the Real-Time R.T.-PCR was performed on the ABI Prism 7700 Sequence Detection System using the TaqMan EZ R.T.-PCR Kit from (Perkin-Elmer Applied Biosystems®).
• R.T.-PCR was optimized for the quantification of the 5' IRES of HCV RNA by using the Taqman technology'(Roche Molecular Diagnostics Systems) similar to the technique previously described ( Martell et al., 1999. J. Clin. Microbiol. 37: 327-332 ).
• the system exploits the 5'-3' nucleolytic activity of AmpliTaq DNA polymerase.
• the method utilizes a dual-labeled fluorogenic hybridization probe (PUTR Probe) that specifically anneals to the template between the PCR primers (primers 8125 and 7028).
• the 5' end of the probe contains a fluorescent reporter (6-carboxyfluorescein [FAM]) and the 3' end contains a fluorescent quencher (6-carboxytetramethylrhodamine[TAMRA]).
• FAM reporter's emission spectrum was suppressed by the quencher on the intact hybridization probe.
• Nuclease degradation of the hybridization probe releases the reporter, resulting in an increase in fluorescence emission.
• the ABI Prism 7700 sequence detector measures the increase in fluorescence emission continuously during the PCR amplification such that the amplified product was directly proportion to the signal.
• the amplification plot was analysed early in the reaction at a point that represents the logarithmic phase of product accumulation.
• a point representing a defined detection threshold of the increase in the fluorescent signal associated with the exponential growth of the PCR product for the sequence detector was defined as the cycle threshold ( C T ).
• C T values are inversely proportional to the quantity of input HCV RNA; such that under identical PCR conditions, the larger the starting concentration of HCV RNA, the lower the C T .
• a standard curve was created automatically by the ABI Prism 7700 detection system by plotting the C T against each standard dilution of known HCV RNA concentration.
• Total cellular Huh-7 RNA was also incorporated with each dilution (50 ng /5 ⁇ L). 5 ⁇ L of each reference standard (HCV Replicon + Huh-7 RNA) was combined with 45 ⁇ L of Reagent Mix, and used in the Real-Time R.T.-PCR reaction.
• the Real-Time R.T.-PCR reaction was set-up for the experimental samples that were purified on RNeasy 96 -well plates by combining 5 ⁇ L of each total cellular RNA sample with 45 ⁇ L of Reagent Mix.
• Reagents and Materials Product COMPANY Catalog # Storage TaqMan EZ R.T.-PCR Kit PE Applied Biosystems N808-0236 -20°C MicroAmp Optical Caps PE Applied Biosystems N801-0935 R.T. MicroAmp Optical 96-Well Reaction Plate PE Applied Biosystems N801-0560 R.T.
• Reagent Mix preparation Component Volume for one sample ( ⁇ L) Volume for One Plate ( ⁇ L) (91 samples + Dead Volume) Final conc.
• NTC No Template Controls
• RNA copy number was normalized (based on the RiboGreen RNA quantification of the total RNA extracted from the cell culture well) and expressed as genome equivalents / ⁇ g of total RNA [g.e./ ⁇ g].
• RNA copy number [g.e./ ⁇ g] from each well of the cell culture plate was a measure of the amount of replicating HCV RNA in the presence of various concentrations of inhibitor.
• the % inhibition was calculated with the following equation: 100 - g . e / ⁇ g inh ⁇ g . e / ⁇ g ctl ⁇ 100 .
• the compounds of this invention are evaluated in the preceding enzymatic and cell based assays, the compounds are found to be highly active.
• the specificity assays used to evaluate the selectivity of this compound are described in WO 00/09543 .
• the compounds of formula 1 were found to be selective in that they do not show significant inhibition (no measurable activity at concentrations up to 30 ⁇ M) in the Human Leukocyte Elastase and Cathepsin B assays.
• the present invention comprises compounds that show pharmacokinetic properties such as detectable plasma levels in the rat at 1 hour and 2 h after an oral dose of 5 mg/kg.
• an in vivo oral absorption screen is used to determine plasma levels of test compounds in a rat after oral administration:
• Each "cassette” contains 3-4 compounds at 5 or 4 mg/kg for each compound.
• the cassettes were prepared as an oral suspension in 0.5% aqueous methylcellulose and 0.3% of polyoxyethylene (20) sorbitan monooleate (Tween-80). The dosing volume was 10 mL/kg via oral gavage.
• Plasma samples were collected at 1 and 2 h post-dosing from the 2 rats and pooled for extraction and analysis.
• Plasma samples at 1 and 2 h, blank plasma, blank plasma spiked with all the compounds at 0.5 ⁇ M of each, are extracted by the solid phase extraction method. Samples were analyzed by HPLC and HPLC/MS for comparison purpose. Plasma concentrations are estimated based on the single concentration of 0.5 ⁇ M standard.

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